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PubMedDate not supplied

Abnormal action-potential bursts and synchronized, GABA-mediated inhibitory potentials in an in vitro model of focal epilepsy.

Focal, freeze-induced lesions were made in isolated hemispheres of turtle cerebral cortex in vitro, permitting the investigation of epileptiform discharges in a preparation with preserved intracortical circuitry. Freeze lesions resulted in interictal discharges and occasional ictal-like events. The interictal discharges were dependent upon activation of non-NMDA excitatory amino acid receptors and were affected by but did not require NMDA receptor activation. Voltage clamp and current clamp recordings revealed abnormal bursts of low-amplitude action potentials in 36% of recorded neurons, while large, repetitive inhibitory potentials, mediated by GABAA receptors, were recorded in 90% of the neurons. Thus, prominent findings in this model include abnormalities of both excitatory and inhibitory activity. Since these changes in neuronal excitability resulted from a localized physical injury, they may resemble the changes that occur in acute posttraumatic epilepsy.

Action Potentials↗
PubMedDate not supplied

Trophoblast invasion of spiral arteries: a novel in vitro model.

Extravillous trophoblasts invade the uterine wall (interstitial invasion) and the spiral arteries (endovascular invasion), replacing the cells of the vessel wall and creating a high-flow low-resistance vessel. We have developed a novel model to allow the interactions between the invading trophoblast cells and the cells of the spiral artery to be directly examined. Unmodified (non-placental bed) spiral arteries were obtained from uterine biopsies at caesarean section. Fluorescently labelled trophoblasts were seeded on top of artery segments embedded in fibrin gels (to study interstitial invasion) or perfused into the lumen of arteries mounted on a pressure myograph (to study endovascular invasion). Trophoblasts were incubated with the vessels for 3-5 days prior to cryo-sectioning. Both interstitial and endovascular interactions/invasion could clearly be detected and a comparison of the extravillous trophoblast cell line, SGHPL-4 and primary first trimester cytotrophoblasts showed both to be invasive in this model. This novel method will prove useful in an area where in vitro studies have been hampered by the lack of suitable models directly examining cellular interactions during invasion.

Adult↗
PubMed2005

In vitro model for evaluating the effect of child formula toothpastes on artificial caries in primary dentition enamel.

PURPOSE: To determine if the in vitro 10-day pH-cycling model used for permanent teeth could be utilized to evaluate de/remineralization effects, on the enamel of primary teeth, of child formula toothpastes. METHODS: Sound extracted primary anterior teeth were coated with nail varnish, leaving a 1 mm-wide window prior to being placed in a demineralizing solution to produce artificial carious lesions in enamel. The teeth were subsequently cut longitudinally into 100 microm thick sections. The 72 specimens were randomly assigned to six groups. In Set 1 contained the specimens in Group a, which were treated with a pea-sized (0.32 g) quantity of non-fluoride toothpaste (First Teeth), and Groups b and c which were treated with half and pea-sized (0.16 g) quantities of fluoridated toothpaste (Colgate), and cycled for 10 days. The specimens in Set 2 (Groups A, B and C) were similarly treated but subjected to 7-day pH-cycling. RESULTS: After Day 8 the lesions in Set 1 extended into dentin and so could not be evaluated. Polarized light microscopy and microradiography were used to evaluate the lesions subjected to 7-day pH-cycling. Lesions in Groups A and B increased in depth and area by approximately 50%, while those in Group C increased in depth and area by 20%; however, no statistically significant differences occurred between the groups. If this model pH cycling is to be used for primary teeth, a reduction from 10 to 7 days in length should be considered.

Cariostatic Agents↗
PubMed2004

The role of topotecan for extending the platinum-free interval in recurrent ovarian cancer: an in vitro model.

OBJECTIVE: Topotecan, a novel topoisomerase-I inhibitor, is an active agent of second-line chemotherapy for extending the platinum-free interval (PFI) and improving the chances of a response to platinum in recurrent ovarian cancer patients. The aim of this study was to understand the molecular mechanism of topotecan-based second-line chemotherapy through an in vitro cell culture model and to gain clinical insight into sequencing issues for second-line treatment with novel agents versus retreatment with platinum. STUDY DESIGN: The human ovarian cancer cell line A2780 and the cisplatin resistance cell line A2780-CR were separately seeded in 6-well cell culture plates and then exposed to multiple concentrations of cisplatin plus paclitaxel or topotecan for 7 days. Surviving cells were recovered and cultured in drug-free media for 3 weeks and then replated in a 96-well microtiter plate. The LD(50) for these cells was determined by a cytotoxic MTT assay after exposure to multiple clinically relevant concentrations of cisplatin or topotecan. Surviving cells were cultured in drug-free media for an additional 4 weeks at which time the LD(50) was reassessed for each cell population by a second MTT assay. Using RT-PCR and Northern blot hybridization to measure mRNA expression, the molecular profile of these cells in terms of resistance was evaluated for the multidrug-resistant gene (MDR-1), multidrug-resistant protein (MRP), Topoisomerase-I, and beta-Actin. RESULTS: The LD(50) to cisplatin was unchanged in A2780-CR cells treated by topotecan. Those A2780-CR cells originally exposed to higher concentrations of cisplatin became more resistant to cisplatin in the MTT assays, while those A2780-CR cell lines treated with a combination of lower cisplatin concentrations and paclitaxel became more sensitive to cisplatin in the MTT assay (P < 0.01). The second MTT assay demonstrated that the LD(50) for cisplatin in every cell line decreased significantly after a 4-week drug-free interval (P < 0.01). There was no difference in the mRNA expression for MRP or topoisomerase-I regardless of cell line, or type or concentration of chemotherapeutic exposure. The mRNA for MDR-1 was uniquely overexpressed in the cisplatin-resistant cell line A2780-CR9 initially treated with low doses of cisplatin and paclitaxel, but was not amplified in A2780 (P < 0.01). CONCLUSIONS: The acquired resistance to cisplatin in A2780 is potentially due to P-glycoprotein-mediated multidrug resistance. This acquired resistance to cisplatin is an unstable phenotype in that some cell populations become sensitive after a drug-free interval and topotecan treatment. This reversal of resistance, however, does not appear to be simply due to loss of MDR-1 expression. While in vivo confirmation is required, agents with novel mechanisms of action offer a strategy to extend the platinum-free interval and thereby improve survival in patients with recurrent ovarian cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1999

An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells.

AIMS/BACKGROUND: One function of Kupffer cells is the phagocytosis of nonviable hepatocytes. Our aims were to develop a model for phagocytosis of damaged hepatocytes by rat Kupffer cells in vitro, and to characterise prostaglandin E2 (PGE2), prostacyclin (PGI), and tumour necrosis factor-alpha (TNF) production in this model. METHODS: Kupffer cells were incubated alone or with damaged hepatocytes for up to 18 h, then washed and cultured for up to 66 h. To compare mediator responses produced during inert particle phagocytosis, Kupffer cells were also incubated with latex beads. RESULTS: Phagocytic uptake of hepatocyte debris was confirmed in at least 50% of Kupffer cells. A dissociation between TNF and PGI responses was found for both latex beads and damaged hepatocytes, such that a TNF secretory response was not triggered by either stimulus whereas PGI production was increased for both. Although phagocytosis of beads increased PGE2 production, phagocytosis of hepatocytes did not. CONCLUSIONS: Phagocytosis of damaged hepatocytes by Kupffer cells results in the production of PGI but not PGE2 or TNF.

Animals↗
PubMed2005

Extracellular proteases of Acanthamoeba castellanii (encephalitis isolate belonging to T1 genotype) contribute to increased permeability in an in vitro model of the human blood-brain barrier.

OBJECTIVES: Granulomatous amoebic encephalitis (GAE) is a serious human infection with fatal consequences, however, the pathogenic mechanisms associated with this disease remain unclear. Several lines of evidence suggest that haematogenous spread is a prerequisite for Acanthamoeba encephalitis but it is not clear how circulating amoebae cross the blood-brain barrier to gain entry into the central nervous system. Objectives of this study were to determine the effects of Acanthamoeba on the permeability of an in vitro blood-brain barrier model and factors contributing to these changes. METHODS: Using human brain microvascular endothelial cells, an in vitro blood-brain barrier model was constructed in 24-well Transwell plates. Acanthamoeba (GAE isolate belonging to T1 genotype) or its conditioned media were used to determine permeability changes. Zymography assays were performed to characterise Acanthamoeba proteases. In addition, the ability of Acanthamoeba to bind brain microvascular endothelial cells was determined using adhesion assays. RESULTS: We observed that Acanthamoeba produced an increase of more than 45% in the blood-brain barrier permeability. Acanthamoeba-conditioned media exhibited similar effects indicating Acanthamoeba-mediated blood-brain barrier permeability is contact-independent. Prior treatment of conditioned media with phenylmethyl sulfonyl fluoride (PMSF, serine protease inhibitor), abolished permeability changes indicating the role of serine proteases. Of interest, methyl alpha-d-mannopyranoside inhibited Acanthamoeba binding to human brain microvascular endothelial cells but had no effect on Acanthamoeba-mediated blood-brain barrier permeability. Zymography assays revealed that Acanthamoeba produced two major proteases, one of which was inhibited by PMSF (serine protease inhibitor) and the second with 1,10-phenanthroline (metalloprotease inhibitor). CONCLUSIONS: We have for the first time shown that Acanthamoeba produces human brain microvascular endothelial cells permeability, which can be blocked by PMSF. A metalloprotease of approx. molecular weight of 150 kDa is produced by A. castellanii (GAE isolate belonging to T1 genotype) and its role in the disease is suggested.

Acanthamoeba castellanii↗
PubMed1990

An in vitro model to study aspects of the pathophysiology of murine rotavirus-induced diarrhoea.

An in vitro system is described and validated for studying transport of solutes and water in both uninfected and rotavirus-infected neonatal mouse intestine. Control intestine exhibited stable water absorption for periods of up to 40 min. Water absorption was temperature-dependent. Na-dependent, and enhanced by glucose-containing perfusion solutions. Theophylline induced net secretion of water by control intestinal tissue. Water transport by rotavirus-infected lower small intestine was significantly depressed as compared to control levels, and rotavirus-infected middle small intestine exhibited net secretion of water. Upper small intestine and colon from infected animals did not differ significantly from control tissues in their ability to transport water. Water secretion by infected middle small intestine was reversed to absorption by glucose-containing solutions.

Animals↗
PubMed1999

Brightness alters Heidelberg retinal flowmeter measurements in an in vitro model.

PURPOSE: The Heidelberg Retinal Flowmeter (HRF), a laser Doppler flowmetry device, has captured interest as a research and clinical tool for measurement of ocular blood flow. Concerns remain about the range and accuracy of the values that it reports. METHODS: An in vitro blood-flow model was constructed to provide well-controlled laminar flow through a glass capillary for assessment by HRF. A change in material behind the glass capillary was used to simulate changing brightness conditions between eyes. RESULTS: Velocities reported by the HRF correlated linearly to true velocities below 8.8 mm/sec. Beyond 8.8 mm/sec, HRF readings fluctuated randomly. True velocity and HRF reported velocities were highly correlated, with r = 0.967 (P < 0.001) from 0.0 mm/sec to 2.7 mm/sec mean velocity using a light background, and r = 0.900 (P < 0.001) from 2.7 mm/sec to 8.8 mm/sec using a darker background. However, a large change in the y-intercept occurred in the calibration curve with the background change. CONCLUSIONS: The HRF may report velocities inaccurately because of varying brightness in the fundus. In the present experiment, a darker background produced an overreporting of velocities. An offset, possibly introduced by a noise correction routine, apparently contributed to the inaccuracies of the HRF measurements. Such offsets vary with local and global brightness. Therefore, HRF measurements may be error prone when comparing eyes. When used to track perfusion in a single eye over time, meaningful comparison may be possible if meticulous care is taken to align vessels and intensity controls to achieve a similar level of noise correction between measurements.

Blood Flow Velocity↗
PubMed2004

Comparison of in vitro models for the prediction of compound absorption across the human intestinal mucosa.

Several in vitro assays have been developed to evaluate the gastrointestinal absorption of compounds. Our aim was to compare 3 of these methods: 1) the bio-mimetic artificial membrane permeability assay (BAMPA) method, which offers a high-throughput, noncellular approach to the measurement of passive transport; 2) the traditional Caco-2 cell assay, the use of which as a high-throughput tool is limited by the long cell differentiation time (21 days); and 3) The BioCoat high-throughput screening Caco-2 Assay System, which reduces Caco-2 cell differentiation to 3 days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations (r = 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat HTS Caco-2 Assay System does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21-day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption.

Biological Assay↗
PubMed1988

The use of renal cortical slices from the Fischer 344 rat as an in vitro model to evaluate nephrotoxicity.

The toxicity of several classical acute nephrotoxicants was evaluated in an in vitro system using renal cortical slices prepared from adult male Fischer 344 rats. Gentamicin, cephaloridine, 4-ipomeanol, potassium dichromate (K2Cr2O7), mercuric chloride (HgCl2), carbon tetrachloride (CCl4), and hexachlorobutadiene (HCBD) were preincubated with slices for 2 hr at 37 degrees C at concentrations ranging from 1 x 10(-6) to 1 x 10(-1) M. Following preincubation, slices were removed, rinsed in medium lacking the nephrotoxicant, and subsequently incubated for 90 min at 25 degrees C for physiological assessment of proximal tubular functions. Using a single preparation, slices were monitored for organic ion accumulation, gluconeogenesis, lipid peroxidation, and total glutathione (reduced and oxidized) concentrations. In addition, preincubation and incubation media were assessed for the presence of renal enzymes originating from brush border, cytosol, and lysosomes. The relative degree of toxicity in this model was consistent with nephrotoxic potency in vivo where HgCl2, K2Cr2O7 greater than HCBD greater than CCl4, cephaloridine greater than gentamicin greater than 4-ipomeanol. Effects on organic ion accumulation, gluconeogenesis, and glutathione concentrations occurred simultaneously for each toxicant. Toxicants produced different effects on enzyme release and malondialdehyde formation. These results suggest that toxicity produced in vitro is representative of in vivo nephrotoxicity and support the further use of this model to evaluate mechanisms of nephrotoxicity.

Acetylglucosaminidase↗
PubMed2005

Evaluation of single versus multiple cryogen spray cooling spurts on in vitro model human skin.

Many commercially available dermatologic lasers utilize cryogen spray cooling for epidermal protection. A previous tissue culture study demonstrated that single cryogen spurts (SCS) of 80 ms or less were unlikely to cause cryo-injury in light-skinned individuals. More recently, multiple cryogen spurts (MCS) have been incorporated into commercial devices, but the effects of MCS have not been evaluated. The aim was to study an in vitro tissue culture model and the epidermal and dermal effects of SCS vs patterns of shorter MCS with the same preset total cryogen delivery time (Deltat(c)) and provide an explanation for noted differences. Four different spurt patterns were evaluated: SCS: one 40-ms cryogen spurt; MCS2: two 20-ms cryogen spurts; MCS4: four 10-ms cryogen spurts; MCS8: eight 5-ms cryogen spurts. Actual Deltat(c) and total cooling time (Deltat(Total)) were measured for each spurt pattern. RAFT tissue culture specimens were exposed to cryogen spurt patterns and biopsies were taken immediately and at days 3 and 7. Actual Deltat(c) was increased while Deltat(Total) remained relatively constant as the preset Deltat(c) of 40 ms was delivered as shorter MCS. Progressively more epidermal damage was noted with exposure to the MCS patterns. No dermal injury was noted with either SCS or MCS. For a constant preset Deltat(c) of 40 ms, delivering cryogen in patterns of shorter MCS increased the actual Deltat(c) and consequently the observed epidermal cryo-injury as compared to an SCS.

Apoptosis↗
PubMed2006

Pharmacodynamic evaluation of tosufloxacin against Streptococcus pneumoniae in an in vitro model simulating serum concentration.

We compared the antibacterial effects and the emergence of resistance to tosufloxacin or levofloxacin for Streptococcus pneumoniae by simulating the serum concentration according to the Japanese clinical regimens using an in vitro pharmacokinetic-pharmacodynamic model. For quinolone-susceptible strain ATCC49619, tosufloxacin showed bactericidal activity, given that both the AUC(0-24h)/MIC ratios at the dosage of 150 mg t.i.d. and 300 mg b.i.d. of tosufloxacin tosilate were 138 and 193, and the C(max)/MIC ranges were 7.93-10.2 and 15.9-17.6, respectively, which were greater than those of levofloxacin (100 mg t.i.d. and 200 mg b.i.d.). The greater area above the killing curves (AAKCs) or shorter time to achieve 99.9% killing (99.9% KT) in both models of tosufloxacin than those of levofloxacin was related to their larger AUC(0-24h)/MIC and C(max)/MIC. Exposure of only 100 mg t.i.d. of levofloxacin led to outgrowth of the parC mutants, which were twofold less susceptible to levofloxacin than the parent strain. Neither of the tosufloxacin tosilate regimens resulted in isolation of resistant mutants of this strain. For the parC mutant strain D-3197, both the AUC(0-24h)/MIC and C(max)/MIC ratios of tosufloxacin were greater than those of levofloxacin, which resulted in comparable or better bactericidal activity as compared to those of levofloxacin. However, both fluoroquinolones and both regimens led to outgrowth of resistant mutants, which possessed a mutation in gyrA in addition to parC. In conclusion, tosufloxacin is superior to levofloxacin in bactericidal activity against S. pneumoniae in the Japanese clinical regimens, especially in the quinolone-susceptible strain, without emergence of resistant subpopulations.

Anti-Bacterial Agents↗
PubMed1993

Dose ranging and fractionation of intravenous ciprofloxacin against Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro model of infection.

The effect of dose or dose interval on the pharmacodynamics of simulated high-dose intravenous ciprofloxacin therapy on infection due to Pseudomonas aeruginosa and Staphylococcus aureus was studied in an in vitro hollow-fiber model of infection. Simulated doses of 1,200 mg of ciprofloxacin per day as either 400 mg every 8 h or 600 mg every 12 h against P. aeruginosa resulted in selection of ciprofloxacin-resistant bacteria. The results with one test strain that was isolated from a patient prior to administration of intravenous ciprofloxacin demonstrated selection of a gyrA mutant in the model, as had occurred in vivo. A single 1,200-mg dose every 24 h did not select for bacterial resistance; however, breakthrough regrowth of ciprofloxacin-susceptible bacteria occurred. Dosages of 400 or 600 mg of ciprofloxacin every 12 h effectively reduced bacterial counts of one strain each of methicillin-susceptible or -resistant S. aureus, with no bacterial resistance detected at the end of experiment; in contrast, 200 mg every 12 h resulted in bacterial regrowth due to the selection of drug-resistant bacteria. These data show the need for high-dose intravenous ciprofloxacin, particularly with regimens producing high peak levels, for treatment of infections where selection for bacterial resistance is a clinical problem.

Ciprofloxacin↗
PubMed2001

In vitro model of a vascular stroma for the engineering of vascularized tissues.

A major problem for the in vitro engineering of larger tissue equivalents like those required in reconstructive surgery is the lack of solutions for sufficient nutrition and oxygenation. The starting point of our investigation was the question of whether the principles of in vitro angiogenesis can be applied and utilized for tissue engineering. A soft tissue model was developed, consisting of human adipose tissue stromal cells and umbilical vein endothelial cells in a fibrin-microcarrier scaffold. Capillary-like structures were visualized using UEA-I-lectin labelling and confocal laser scanning microscopy. Length of capillary-like structures was measured in an image analysis system. Under serum-free culture conditions, maintenance of capillary-like structures was significantly increased in comparison to serum-containing cultures. The application of vascular endothelial growth factor (VEGF) resulted in a high initial angiogenic response; long-term stabilization of capillary-like structures could not be achieved, however supplementation with IGF-1 resulted in the highest values and the slightest decrease in length of capillary-like structures, so that the results could be interpreted as an improved stabilization.

Adipose Tissue↗